Lipopolysaccharide抗血小板凝集作用之機轉探討
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Lipopolysaccharide抗血小板凝集作用之機轉探討. Lipopolysaccharide (LPS)為格蘭氏陰性菌(Gram-negative bacteria)細 胞外 壁上的一種脂多醣體,其分子量約為1×105 ~ 9×105 Da之間。在 此類細菌感染而致病的 原因中,主要是其分子上的LPS扮演決定性的角 色。譬如LPS對全身循環系統的影響,如引起低血壓、腎臟皮質壞死、影 響血球的數目、使循環中血小板數目減少、造成瀰漫性血管 內凝血( DIC)、甚至引起敗血性休克而最後導致死亡。其中LPS對血小板的影響如

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Lipopolysaccharide抗血小板凝集作用之機轉探討

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Lipopolysaccharide

Lipopolysaccharide抗血小板凝集作用之機轉探討

  • Lipopolysaccharide (LPS)為格蘭氏陰性菌(Gram-negative bacteria)細

  • 胞外 壁上的一種脂多醣體,其分子量約為1×105 ~ 9×105 Da之間。在

  • 此類細菌感染而致病的 原因中,主要是其分子上的LPS扮演決定性的角

  • 色。譬如LPS對全身循環系統的影響,如引起低血壓、腎臟皮質壞死、影

  • 響血球的數目、使循環中血小板數目減少、造成瀰漫性血管 內凝血(

  • DIC)、甚至引起敗血性休克而最後導致死亡。其中LPS對血小板的影響如

  • 何? 其機 轉又為何? 眾說紛云,尚無明確的答案,這也是本篇論文所要

  • 探討的目的。 在人 類及兔子的血小板懸浮液中,發現LPS能隨著

  • 濃度和時間的增加而有效地抑制由collagen (5 mg/ml)、U46619 (1

  • mM)、thrombin (0.5 U/ml)、ADP (10 mM)、PAF (0.8 ng/ml)所 引起的

  • 血小板凝集及ATP釋放反應。在人類富含血小板血漿中亦有相同的抑制作

  • 用。因LPS (100, 200和500 mg/ml)能依劑量相關性(dose-dependent)

  • 的抑制不同活化劑所引起的血 小板凝集反應;所以,我們懷疑LPS是否

  • 是作用在血小板凝集的最後共同路徑上;也就是 纖維蛋白原(

  • fibrinogen)與血小板細胞膜上的glycoprotein IIb/IIIa complex的結合

  • , 進而抑制血小板凝集反應。實驗發現LPS並無法影響FITC標定的

  • triflavin結合到細胞膜上 glycoprotein IIb/IIIa complex;

  • triflaflavin是一種蛇毒蛋白,已證實為一種glycopr otein IIb/IIIa

  • complex的拮抗劑。因此,本實驗證實LPS抑制活化劑所引起的血小板凝

  • 集反應,可能與抑制glycoprotein IIb/IIIa complex無關。另外LPS

  • (100, 200和500 mg /ml)能明顯地抑制collagen所引起的

  • phosphoinositides (PI) 的分解,且亦能抑制colla 所引起的血小板細

  • 胞內鈣離子的增加,但對collagen所引起的thromboxane B2的形成 ,則

  • 無明顯抑制作用。在偵測細胞膜流動性的實驗中,Lg/ml)會明顯減少

  • diphenylhexatr iene (DPH)標定到細胞膜的程度,此暗示著LPS會干擾

  • 血小板細胞膜的流動性;在測量lac tate dehydrogenase (LDH)的實驗

  • 方面,LPS (100, 200和500 mg/ml)亦會有意義的增加 血小板細胞膜外

  • 的LDH (約7 ~ 9 %)含量,此點暗示著LPS可能會經由改變細胞膜的流動性

  • 進而促使些微的LDH釋放。再者,在探討PKC pathway時,我們發現LPS

  • (200和500 mg/ml) 與血小板懸浮液溫浴60分鐘後,會部分抑制PKC的活

  • 化劑如PDBu所引起的血小板凝集反應(約30 %);另外,LPS也會有意義的

  • 抑制47 kDa protein的磷酸化反應。此外,LPS (200和 500 mg/ml)不會

  • 有意義的增加血小板內cyclic AMP的含量,但能提高cyclic GMP在血小板

  • 中的濃度,此點暗示著是否LPS是藉由促使血小板NO的形成進而增加

  • cyclic GMP的含量; 利用chemiluminesence的方法進一步偵測LPS對血

  • 小板中NO的影響,實驗結果亦發現LPS會明顯的增加NO的濃度。綜合以上

  • 的結果,LPS對血小板的抑制作用機轉可能有以下二點: (一) LPS直接

  • 改變了血小板細胞膜的流動性,進而影響了一些嵌在細胞膜上的蛋白質的

  • 作用,如phospholipase C (PLC),使得phosphoinositides (PI)分解後

  • 產生inositol-1,4, 5-trisphosphate (IP3)與1,2-diacylglycerol

  • (DG)的含量減少,IP3的減少會降低細胞 內鈣離子的濃度,進而抑制血

  • 小板的凝集及釋放作用,而DG減少則會進一步抑制47 kDa p rotein

  • phosphorylation。(二) LPS活化血小板內的NO synthetase (NOS),進而

  • 促進NO 形成,接著活化了guanylate cyclase,使cyclic GMP的量增加

  • ,接著抑制phospholipase的活性,減少PI的分解,再使得細胞內鈣離子

  • 濃度降低,而抑制血小板的凝集反應。


Lipopolysaccharide

Mechanisms involved in the antiplatelet activity of lipopolysaccharide

  • Lipopolysaccharide (LPS) was originally known as endotoxin andsat on the out er membrane of the Gram-negative bacteria. Themoleculer weight of LPS was abo ut 1×105 ~ 9×105 Da. A singleinjection of LPS can produce hypotension, rena l corticalnecrosis, changes in numbers of peripheral blood cells,thrombocyto penia, disseminated intravascular coagulation(DIC), and septic shock. However , the exact machanism is stillunclear and requires further characterization.I n our studies,we found that LPS can dose-dependently (100 ~ 500 mg/ml) and time-dependently (10 ~ 60 min) inhibit platelet aggregationinduced by collagen (5 mg/ml), U46619 (1 mM), thrombin (0.5 U/ml), ADP (10 mM), and PAF (0.8 ng/ml ) in human and rabbitplatelet suspensions. The similar results were also obta inedin the preparation of platelet-rich plasma (PRP). This may implythat whe ther LPS inhibits platelet aggregation throughdirectly interfering with fibri nogen binding to fibrinogenreceptor associMeasurement of the platelet membran e fluidity,we found that LPS was capable of direct interaction withplatelet membrane fluidity tragged with diphenylhexatriene(DPH). In LDH assay, LPS ind uced a slight release (7 ~ 9 %) oflactate dehydrogenase (LDH) from platelet c ytosol in humanplatelets. In protein kinase C experiments, LPS (200 and 500 mg/ml) also significantly inhibited platelet aggregation anddecreased a 47 kDa protein phosphorylation induced by PDBu(0.05 mM), a PKC activator. On the ot her hand,Therefore, basedon the above observations, we suggested that there a re twopossibilities involving in the antiplatelet activity of LPS: (1)LPS in fluenced the platelet membrane fluidity is the primarymechanism, followed by the inhibition of phospholipase Cactivity, thereby leading to the inhibition of [Ca2+]imobilization and platelet aggregation induced by agonists. (2)LPS induced NO formation through directly activation of NOsynthetase, resulting f inally in increase the cGMP levels inhuman platelets


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